I present a 5-year career development plan that seeks to understand the role of epigenetic changes in normal hematopoietic development and MLL-rearranged leukemia, while establishing an academic career in Hematopoietic Stem Cell Transplantation. I will build on my strong foundation in molecular biology and clinical hematology/oncology to develop expertise in epigenetic regulation, hematopoietic stem cell, and leukemia biology under the leadership of Dr. Stuart Orkin and Dr. Scott Armstrong. Dr Orkin is a pioneer and world leader in hematopoietic development and genome wide genetic, epigenetic and proteomic studies. Dr Armstrong is a world leader in genomic approaches and epigenetic changes in MLL rearranged leukemias. In the recent years, epigenetic modifications have started to be recognized as a dynamic and finely regulated processe involved in transcriptional control and major cell fate decisions. The histone methyl transferase Dot1l, which methylates histone 3 at lysine 79 (H3K79), has recently been implicated as a crucial component in mixed lineage leukemia (MLL) gene rearranged leukemias. Unpublished data also point to a fundamental role in early hematopoietic development. We have generated a conditional loss of function mouse model for Dot1l, and we will use this model to directly address the role of Dot1l in hematopoiesis and leukemia. In specific aim 1 we will characterize the hematopoietic phenotype of Dot1l inactivation during hematopoietic development and steady state adult hematopoiesis. We will assess embyonic hematopoietic development, stem and progenitor cell frequencies, self-renewal and differentiation in vitro and in vivo through a series of genetic and transplantation experiments. We will also investigate how loss of H3K79 methylation affects other chromatin modifications in hematopoietic stem cells on a genome wide level. In specific aim 2 we will test the hypothesis that mis-recruitment of DOT1L through MLL-fusion proteins is a fundamental step in MLL-mediated leukemogenesis, and required for leukemia cell survival. To this end, we will assess the development and maintenance of leukemia induced by the common fusions Mll-Af4 and Mll-Af9 in our conditional Dot1l knockout mouse model. We will conduct DOT1L sh-RNA knock down experiments in human MLL-rearranged leukemia cell. In addition, we plan to utilize our models to validate potential small molecule inhibitors for DOT1L in collaboration with Dr. Stuart Schreiber. Execution of these aims will elucidate fundamental mechanisms of chromatin regulation in benign and malignant hematopoiesis.